Thrombopoietin (Tpo) is the primary physiologic regulator of megakaryocyte and platelet development through the Tpo receptor mpl. Recently we have made the novel and serendipitous observation that Tpo is an extraordinarily potent cardioprotective agent. These original observations may cause a paradigm shift in how we view the mechanisms and biological effects of Tpo. Thus the overall objective of this proposal is to define the intracellular signaling pathways by which Tpo confers early and delayed cardioprotection. Whereas there is a substantial body of literature regarding the mpl receptor composition, distribution and signaling in platelet function, there is no information about the pathway by which binding of Tpo to mpl confers cardioprotection and how this pathway is recruited by hypoxia in the heart. We hypothesize that activation of the Tpo receptor triggers pro-survival pathways and suppresses apoptosis to confer early and delayed cardioprotection by opening of mitochondrial KATp channels and/or mitochondria! KCa channels. Specifically we shall: 1) Determine the pathways that confer early cardioprotection by Tpo. We shall determine whether Tpo signals through its receptor (mpl) to activate the pro-survival kinase signaling cascade: JAK/STAT, Ras, Raf, phosphatidylinositol-3-OH kinase (PI3K)/Akt, PKC, JNK, p38 MARK and p42/44 MARK. We shall determine whether protection by Tpo given immediately following reperfusion acts by activating the pro-survival RISK pathway and whether Tpo protects the chronically hypoxic heart. 2) Determine the relative importance of the mitochondrial KATP channel, KCa channel and the sarcolemmal KATP channel in early cardioprotection produced by Tpo. We shall determine if Tpo signaling in early cardioprotection is mediated by opening of mitochondrial KATP and KCa channels. We shall measure mitochondrial membrane potential, respiration and ATP synthesis in mitochondria isolated from Tpo-treated hearts to determine the relationship between KATP channels to mitochondrial function. 3) Determine the effect of chronic hypoxia on the expression of Tpo and the Tpo receptor (mpl) in rabbit and human heart. We shall utilize our model of chronic hypoxia to determine in both infant human and rabbit hearts whether adaptation to chronic hypoxia increases expression of message and protein for mpl and circulating levels of platelets. 4) Determine the mediators of delayed cardioprotection produced by Tpo. We shall determine if "NO acts as a mediator of delayed cardioprotection by Tpo. We shall then determine if mediation of delayed cardioprotection by Tpo occurs by opening sarcolemmal and/or mitochondrial KATP channels. The results of these studies are expected to have an impact on our understanding of the biology of Tpo in heart and may lead to the development of new therapeutics for the treatment of myocardial infarction.